Growth factor isoform

ABSTRACT

An isolated VEGF polypeptide having anti-angiogenic activity, said polypeptide including the amino acid sequence of SEQ. ID NO. 1, or variants thereof.

The present invention relates to novel VEGF isoforms and their use asanti-angiogenic, anti-vasodilatory, anti-permeability andanti-proliferative agents and inhibition of such isoforms in conditionsin which their expression in excess may be associated with diseasestates.

In order for tissue to grow it needs to develop and maintain an activeand highly efficient blood supply from the surrounding vasculature. Thisprocess (angiogenesis) is necessary for progression and survival oftumours, development of rheumatoid arthritis, psoriasis, proliferativeeye disease and a host of other pathologies. A number of new anti-tumourtherapies are being developed that target this novel vasculature, or thegrowth factors that produce it. This is of particular interest in cancerresearch. This novel approach to cancer therapy contrasts withtraditional chemotherapeutic strategies which target cell proliferation.The advantage of this approach lies in the distinction that angiogenesisis not an essential component of normal physiology (except in thedevelopment of the corpus luteum, endometrium and placenta inpre-menopausal women), and therefore anti-angiogenesis is not as toxicas anti-proliferative therapies, which also affect hair growth, normalgastrointestinal physiology, skin epidermis and many other normalaspects of physiology. The therapeutic dose of anti-angiogenics istherefore predicted to be well below the most tolerated doses, unlikeanti-proliferative drugs. Tumours develop and maintain their new bloodsupply by secreting endothelial cell-specific growth factors, a numberof which have been isolated in the last few years. One of these isVascular Endothelial Growth Factor (VEGF), a naturally secreted proteinthat stimulates angiogenesis, vasodilatation and increased vascularpermeability in vivo.

Vascular endothelial growth factor (VEGF) is a 32-42 kDa dimericglycoprotein which mediates vasodilatation, increased vascularpermeability and endothelial cell mitogenesis. Differential exonsplicing of the VEGF gene results in three main mRNA species which codefor three secreted isoforms (subscripts denote numbers of amino acids):VEGF₁₈₉, VEGF₁₆₅, and VEGF₁₂₁. A number of minor splice variants havebeen described (VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₅ and VEGF₁₄₈) but theirimportance remains uncertain (FIG. 1). Each isoform has distinctproperties and patterns of expression (Ferrara et al (1997) Endocr. Rev.18, 4-25; Houck et al (1991) Mol. Endocrinol. 5, 1806-1814; Poltorak etal (1997) J. Biol. Chem. 272, 7151-7158; Simon et al (1995) Am. J.Physiol. 268, 240-250; Brown et al (1992) Kidney Int. 42, 1457-1461;Park et al (1993) Mol. Biol. Cell 4, 1317-1326; Kevt et al (1996) J.Biol. Chem. 271, 7788-7795; Jingling et al (1999) Invest Opthalmol VisSci 40(3):752-9; Plouet et al (1997) J. Biol. Chem 272, 13390-13396; andWhittle C et al Clin Sci (1999) 97, 303-312);. The various molecularforms of VEGF share a common amino-terminal domain consisting of 110amino acids, but differ in the length of the carboxyl-terminal portionand the final 6 amino acid residues (coded for by exon 8) are identicalin all isoforms previously described except VEGF₁₄₈.

VEGF is known to promote vascular endothelial cell proliferation andangiogenesis, which are important components of a variety ofpathologies, including tumour growth and metastasis, rheumatoidarthritis, atherosclerosis and arteriosclerosis (Celleti et al Nature2001; 7. 425-9; Lemstrom et al 2002, 105. 2524-2530), neointimalhyperplasia, diabetic retinopathy and other complications of diabetes,trachoma, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, trachoma haemangiomata, immune rejection oftransplanted corneal tissue, corneal angiogenesis associated with ocularinjury or infection, psoriasis, gingivitis and other conditions known tobe associated with angiogenesis and/or chronic inflammation. Cornealangiogenesis associated with ocular injury or infection may be caused,for example, by Herpes or other viral or microbial infection.

In addition VEGF expression and angiogenesis are increased during fatdeposition in animal models (Fredriksson et al (2000), J. Bio. Chem.275(18): 13802-11; Asano et al (1999) J. Vet. Med. Sci. 61(4):403-9;Tonello et al (1999) FEBS Lett 442(2-3): 167-72; Asano et al (1997)Biochem. J. 328(Pt.1): 179-83), and it is therefore possible thatanti-angiogenic agents may be used for reducing fat deposition and forfat reduction.

VEGF may also mediate pre-eclampsia (Brockelsby et al 1999, Lab Invest79:1101-11). Pre-eclampsia is a condition of pregnancy characterised byhigh blood pressure, persistent excessive swelling of the hands, feet,ankles and sometimes face, and protein in the urine. If not diagnosedand treated quickly, it can lead to eclampsia, where a seizure occurs,coupled with a sharp rise in blood pressure and serious risk of stroke,as well as extreme distress to an unborn baby. Dysregulation ofvasoconstriction/vaso-dilatation is known to be an important componentof pre-eclampsia. Despite the fact that VEGF has been implicated inpre-eclampsia, one paradox is as yet unexplained; pre-eclampsia isassociated with hypertension and vasoconstriction but VEGF₁₆₅ is a wellknown vasodilator.

VEGF also increases endothelial permeability which is an importantcomponent of a different variety of conditions including angiogenesisrelated oedema (i.e. in tumours), septicaemic/endotoxaemic shock,nephrotic syndrome, lymphoedema, burns and adult respiratory distresssyndrome (ARDS) and pre-eclampsia as above.

The endothelial proliferative activity of VEGF is mediated by two highaffinity membrane-bound tyrosine kinase receptors: VEGF receptor 1(VEGFR₁, flt-1(fms-like tyrosine kinase-1)); and VEGF receptor 2(VEGFR₂, flk (foetal liver kinase), KDR (Kinase domain containingreceptor)). These receptors are expressed by vascular endothelial cells.

It is believed that normally both of the VEGF receptors are stimulatedby a homodimer of VEGF monomers interacting with a homodimer of receptormolecules.

In view of the implied role of VEGF-mediated receptor stimulation invarious diseases, there is considerable interest in developinginhibitors that would interfere with or modulate the interaction betweenVEGF and its receptor.

The major obstacle to the development of novel anti-angiogenic drugs inoncology and other specialities, however, has been the difficulty inobtaining angiogenic-specific inhibitors with acceptable half lives,solubility, specificity and immunotolerance.

Tissues are normally in an angiogenesis equilibrium, i.e. growth factorswhich stimulate new vessel growth are balanced by other factors whichinhibit vessel growth (M. L. IruelaArispe and H. F. Dvorak, (1997)Thrombosis and Haemostasis. (78): 672-677). One of the tissues in whichVEGF is normally most highly expressed is in the kidney glomerulus (L.F. Brown, et al., (1992) Kidney Int. (42):1457-61 E. Bailey 1999 J ClinPathol 52, 735-738.). This tissue expresses high levels of VEGF, hasvery high endothelial permeability (an action of VEGF), but has lowlevels of angiogenesis. The reason for the high VEGF expression levelsin kidney is not known but the high permeability of the endothelium andhigh glomerular filtration rate both appear to depend on VEGF (B.Klanke, et al., (1998) Nephrol Dial Transplant, (13):875-850) (C.Whittle, et al., (1999) Clin Sci (Colch). (97):303-12). It is notunderstood why there is no angiogenesis in kidneys in the presence ofsuch high expression of VEGF.

The present inventors have identified a new isoform of VEGF in kidneycells, which is differentially spliced into a previously undescribedexon, exon 9. This novel isoform has been designated VEGF₁₆₅b.

According to a first aspect of the invention, there is provided anisolated VEGF polypeptide having anti-angiogenic activity, saidpolypeptide including the amino acid sequence of SEQ.ID NO.1, orvariants thereof.

This unexpected anti-angiogenic property of the polypeptide of the firstaspect of the present invention contrasts completely with the propertiesof all previously described VEGF isoforms which are pro-angiogenic.

The term “isolated” as used herein means altered from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated”,but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is usedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by anotherrecombinant method is “isolated” even if it is still present in saidorganism, which organism can be living or non-living.

The term “variant(s)” as used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and/or truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A-variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, and/or deletions in any combination. Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. The present invention also includes variants ofeach of the polypeptides of the invention, that is polypeptides thatvary from the references by conservative amino acid substitutions,whereby a residue is substituted by another with like characteristics.Typical conservative amino acid substitutions are among Ala, Val, Leuand Ile; among Ser and Thr; among the acidic residues, Asp and Glu;among Asn and Gln; and among the basic residues Lys and Arg; or aromaticresidues Phe and Tyr. Such conservative mutations include mutations thatswitch one amino acid for another within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Gluand Gln;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

Such conservative variations can further include the following: OriginalResidue Variation Ala Gly, Ser Arg Lys Asn Gln, His Asp Glu Cys Ser GlnAsn Glu Asp Gly Ala, Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Tyr, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr TyrTrp, Phe Val Ile, Leu

The types of variations selected can be based on the analysis of thefrequencies of amino acid variations between homologous proteins ofdifferent species developed by Schulz et al. (Principles of ProteinStructure, Springer-Verlag, 1978), on the analyses of structure-formingpotentials developed by Chou and Fasman (Biochemistry 13:211, 1974 andAdv. Enzymol., 47:45-149, 1978), and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al. (Proc. Natl. Acad.Sci. USA 81:140-144, 1984), Kyte & Doolittle (J. Molec. Biol. 157:105-132, 1981), and Goldman et al. (Ann. Rev. Biophys. Chem. 15:321-353,1986). Particularly preferred are variants in which several, e.g., 5-10,1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto a person skilled in the art.

The term “nucleotide(s)” as used herein generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Polynucleotide(s) include, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions or single- and triple-strandedregions, single- and double-stranded RNA, and RNA that is a mixture ofsingle- and double-stranded regions, hybrid molecules comprising DNA andRNA that can be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. As used herein, the term “polynucleotide(s)” also includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotide(s)” as the term is intended herein.Moreover, DNAs or RNAs comprising unusual bases, such as inosine, ormodified bases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The term“polynucleotide(s)” as used herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

The term “polypeptide(s)” as used herein refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers and to longer chains generally referred to as proteins.Polypeptides.can contain amino acids other than the 20 gene encodedamino acids. “Polypeptide(s)” include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in research literature, and are wellknown to those skilled in the art. It will be appreciated that the sametype of modification can be present at the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide cancontain many types of modification. Modification can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chains,and the amino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or a nucleotide derivative, covalent attachment of a lipidor lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation orglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2^(nd) Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects. pgs1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson. Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides can be branched, or cyclic, with or withoutbranching. Cyclic, branched and non-branched polypeptides can resultfrom post-translational natural processes and can be made by entirelysynthetic methods as well.

Preferably, at least a portion of the polypeptide comprises at least 66%identity to the sequence shown in SEQ.ID NO.1. More preferably, a leasta portion of the polypeptide comprises at least 83% identity to thesequence shown in SEQ.ID NO.1.

Identity, as used herein, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and genomeProjects, Smith, D. W., ed., Academic Press, New York. 1993; ComputerAnalysis of sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press. New jersey, 1994; sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073(1998). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NUH Bethesda, Md. 20894;Altschul, S., et al., J. Mol Biol. 215: 403-410 (1990).

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff & Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparison (along with no penalty for end gaps). In all cases where acomputer program that does not necessarily give the maximized alignmentdiscussed above is used to determine a measure of identity, the defaultparameters are preferred. Parameters for polynucleotide comparisoninclude the following: Algorithm; Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Preferably, the sequence shown in SEQ ID NO. 1, or variants thereof,occurs at the C-terminus of the polypeptide.

Preferably, the polypeptide lacks exon 8. This will result in apolypeptide either lacking or having altered mitotic signalling comparedto polypeptides containing exon 8.

A further aspect of the invention provides an isolated polypeptidehaving anti-angiogenic activity, the polypeptide comprising the sequenceshown in SEQ. ID NO. 3, and variants thereof.

A further aspect of the invention provides a nucleotide sequence capableof encoding a polypeptide according to a first or second aspect of theinvention.

A further aspect of the invention provides an isolated nucleotidesequence encoding a polypeptide having anti-angiogenic activity, thenucleotide sequence comprising the sequence shown in SEQ.ID No.2, orvariants thereof.

A further aspect of the present invention provides an isolatednucleotide sequence encoding a polypeptide having anti-angiogenicactivity, the nucleotide sequence comprising the sequence shown in SEQ.ID NO. 4, or variants thereof.

A further aspect of the present invention provides a polynucleotidecomprising a nucleotide sequence that hybridises, particularly understringent conditions, to a VEGF,₆₅b nucleotide sequence, such as thenucleotide sequence shown in SEQ. 2.

As used herein the term “stringent conditions” means hybridisationoccurring only if there is at least 83% identity between the sequences.A specific example of stringent hybridisation conditions is overnightincubation at 42° C. in a solution comprising: 50% formamide. 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulphate, and 20 micrograms/ml ofdenatured, sheared salmon sperm DNA, followed by washing thehybridisation support in 0.1×SSC at about 65° C. Hybridisation and washconditions are well known and exemplified in Sambrook, et al., MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring Harbour, N.Y.,(1989), particularly Chapter II therein. Solution hybridisation can alsobe used with the polynucleotide sequences provided by the invention.

A nucleotide sequence according to the invention may be used as ahybridisation probe for RNA, cDNA and genomic DNA to isolate full-lengthcDNAs and genomic clones encoding VEGF₁₆₅b and to isolate cDNA andgenomic clones of other genes that have a high identity, particularlyhigh sequence identity, to the VEGF₁₆₅b gene. Such probes will generallycomprise at least 15 nucleotide residues or base pairs, and can have atleast 18 nucleotide residues or base pairs.

Preferably, the polypeptide and nucleotide sequence of the presentinvention is a mammalian sequence, such as a primate, rodent, bovine orporcine sequence. More preferably, the sequence is derived from a humansequence. The nucleotide sequence may include, for example, unprocessedRNAs, ribozyme RNAs, hair-pin RNAs for use as interference RNAs, smallinterfering RNAs (siRNAs), mRNAs, cDNAs, genomic DNAs, B-DNAs, E-DNAsand Z-DNAs.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into a host cell may be effected by methods described inmany standard laboratory manuals, such as Davis, et al., BASIC METHODSIN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbour Laboratory Press, ColdSpring Harbour. N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transfection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomyces,cyanobacteria, Bacillus subtilis; fungal cells, such as yeast,Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans andAspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9;animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowesmelanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and viral-derived vectors, for example, vectorsderived from plasmids, from bacteriophage, from transposons, from yeastepisomes, from insertion elements, from yeast chromosomal elements, fromviruses such as baculoviruses, papova viruses, such as SV40, vacciniaviruses, adenoviruses, adeno-associated viruses, fowl pox viruses,pseudorabies viruses, picomaviruses and retroviruses, and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs can contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides or to express apolypeptide in a host can be used for expression in this regard. Theappropriate DNA sequence can be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL, (supra).

For secretion of a translated protein into the lumen of the endoplasmicreticulum, into the periplasmic space or into the extracellularenvironment, appropriate secretion signals can be incorporated into theexpressed polypeptide. These signals can be endogenous to thepolypeptide or they can be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods, including ammoniumsulphate or ethanol precipitation, extraction such as acid extraction,anion or cation exchange chromatography, gel filtration,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, lectinchromatography, preparative electrophoresis, FPLC (Pharmacia, Uppsala,Sweden), HPLC (e.g., using gel filtration, reverse-phase or mildlyhydrophobic columns). Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding proteins can be employed to regenerate an active conformationafter denaturation of the polypeptide during isolation and/orpurification. In vitro activity assays for polypeptides according to thepresent invention include, tyrosine kinase receptor activation assays,endothelial cell proliferation (e.g. thymidine incorporation, cellnumber or BrDU incorporation), cell migration assays (including scratchassays), tube formation, gel invasion assays or pressure or wiremyograph assays. In vivo assays include angiogenesis assays using rabbitcorneal eye pocket, chick chorioallantoic membrane assays, dorsalskinfold chamber assays, functional blood vessel density, blood flow,blood vessel number, tumour implantation assays (syngeneic orheterogeneic), tumour growth or vessel density assays, growth factorinduced assays in hamster cheek pouch, rat, mouse or hamster mesentery,or sponge implant assay (Angiogenesis protcols—Ed. J. Clifford Murray;Humana Press, Totowa, N.J.; ISBN 0-89603-698-7 (part of a Methods inMolecular Medicine series)).

A polypeptide, or polynucleotide comprising a nucleotide sequence,according to the present invention or variants thereof, or cellsexpressing the same can be used as immunogens to produce antibodiesimmunospecific for such polypeptides or nucleotide sequencesrespectively.

A further aspect of the invention provides an isolated VEGF polypeptideor an isolated nucleotide sequence according to a preceding aspect ofthe present invention for use as an active pharmaceutical substance.

The active pharmaceutical substance is preferably used in the treatmentof angiogensis or permeability or vasodilatation-dependent diseaseconditions as detailed above. Preferably angiogenesis-dependent diseaseconditions such as tumour growth and metastasis, rheumatoid arthritis,atherosclerosis, neointimal hyperplasia, diabetic retinopathy and othercomplications of diabetes, trachoma, retrolental fibroplasia,neovascular glaucoma, age-related macular degeneration, trachoma,haemangioma, immune rejection of transplanted corneal tissue, cornealangiogenesis associated with ocular injury or infection, vasculardisease, obesity, psoriasis, arthritis, gingivitis and pre-eclampsia.

A further aspect of the invention provides a use of an isolated VEGFpolypeptide sequence or an isolated nucleotide sequence according topreceding aspects of the present invention for the preparation of apharmaceutical composition for the treatment of angiogenesis-dependentdisease conditions such as previously mentioned.

A further aspect of the invention provides a method for treating orpreventing angiogenesis in a mammalian patient, comprising supplying tothe patient a polypeptide comprising the sequence of a VEGF polypeptideaccording to a previous aspect of the invention.

Preferably, an isolated VEGF polypeptide according to a previous aspectof the invention is capable of heterodimerising with endogenous VEGFthereby preventing or reducing VEGF-mediated cell proliferation or bydirect binding to receptor normally the ligand for endogenous VEGF.

A further aspect of the invention provides a method for treating orpreventing angiogenesis in a mammalian patient, comprising supplying tothe patient a polynucleotide comprising a nucleotide sequence accordingto a previous aspect of the invention.

A further aspect of the invention provides a method for preventing orreducing VEGF-mediated cell proliferation in a mammalian patientcomprising supplying to the patient a polypeptide comprising thesequence of an isolated VEGF polypeptide according to a previous aspectof the invention.

A further aspect of the invention provides a method for preventing orreducing VEGF-mediated cell proliferation in a mammalian patientcomprising supplying to the patient a polynucleotide comprising thesequence of an isolated nucleotide sequence according to a previousaspect of the invention.

A further aspect of the invention provides a method for preventing orreducing VEGF₁₆₅-mediated vasodilatation in a mammalian patientcomprising supplying to the patient a polypeptide comprising thesequence of a VEGF polypeptide according to a previous aspect of theinvention.

A further aspect of the invention provides a polypeptide comprising thesequence of a VEGF polypeptide according to a preceding aspect of theinvention for use in the treatment of VEGF₁₆₅-mediated vasodilatation.

A further aspect of the invention provides a use of a polypeptidecomprising the sequence of a VEGF polypeptide according to a precedingaspect of the invention for the preparation of a pharmaceuticalcomposition for the treatment of VEGF₁₆₅-mediated vasodilatation.

A further aspect of the invention provides a method for preventing orreducing VEGF₁₆₅-mediated vasodilatation in a mammalian patientcomprising supplying to the patient a polynucleotide comprising thesequence of an isolated nucleotide according to a preceding aspect ofthe invention.

A further aspect of the invention provides a polynucleotide comprisingthe sequence of a nucleotide according to a previous aspect of theinvention for use in the treatment of VEGF₁₆₅-mediated vasodilatation.Conditions in which VEGF₁₆₅-mediated vasodilatation is observed include,for example, cancer, psoriasis, arthritis etc.

A further aspect of the invention provides a use of a polynucleotidecomprising a nucleotide sequence according to a preceding aspect of theinvention in the preparation of a pharmaceutical composition for thetreatment of VEGF₁₆₅-mediated vasodilatation.

A further aspect of the invention provides a pharmaceutical compositioncomprising a polypeptide comprising the sequence of a VEGF polypeptideaccording to a previous aspect of the invention and a pharmaceuticallyacceptable diluent.

Preferably, the vasodilatation and/or angiogenesis is associated withhair growth, as seen, for example, in hirsuitism. Any reduction invasodilatation and/or angiogenesis would result in hair loss (Yano et alJ Clin Invest (2001), 107 409-17).

A further aspect of the invention provides a pharmaceutical compositioncomprising a polynucleotide comprising a nucleotide sequence accordingto a previous aspect of the invention and a pharmaceutically acceptablediluent.

A further aspect of the invention provides an antibody raised against aVEGF polypeptide or nucleotide sequence according to a previous aspectof the invention.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCOLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms such as other mammals, can be used to expresshumanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology can be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-VEGF₁₆₅b or fromlibraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, etal., (1992) Biotechnology 10, 779-783). The affinity of these antibodiescan also be improved by, for example, chain shuffling (Clackson et al.,(1991) Nature 352:628).

The above-described antibodies can be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

The polynucleotides, polypeptides and antibodies that bind to orinteract with a polypeptide of the present invention can also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA or polypeptide in cells. For example, an ELISAassay can be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich can inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the anti-angiogenicaction of VEGF₁₆₅b polypeptides or polynucleotides. The method ofscreening can involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising VEGF₁₆₅b polypeptide and alabelled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a VEGF₁₆₅bagonist or antagonist. The ability of the candidate molecule to agonizeor antagonize the VEGF₁₆₅b polypeptide is reflected in decreased bindingof the labelled ligand or decreased production of product from suchsubstrate. Molecules that bind gratuitously, i.e., without inducing theanti-angiogenic effects of VEGF₁₆₅b polypeptide are most likely to begood antagonists. Molecules that,bind well and increase theanti-angiogenic effects of VEGF₁₆₅b are agonists. Detection of theincrease in anti-angiogenic action can be enhanced by using a reportersystem. Reporter systems that can be useful in this regard include butare not limited to colorimetric, labelled substrate converted intoproduct, a reporter gene that is responsive to changes in VEGF₁₆₅bpolynucleotide or polypeptide activity, and binding assays known in theart.

The invention further provides an inhibitor of a VEGF polypeptideaccording to a preceding aspect of the invention, for use in thetreatment of vasoconstriction.

As pre-eclampsia results from poor placental invasion, i.e. a failure ofangiogenesis, and VEGF antibodies have been shown to block the cause ofhypertension in pre-eclampsia (Brockelsby et al Lab Invest 1999;79:1101-11) this is considered by the present inventors to bepotentially caused by excess new variant VEGF₁₆₅b, and thus inhibitionof this new variant VEGF₁₆₅b, is expected to provide a treatment forpre-eclampsia.

It is presently believed that the inhibitor is capable of bindingendogenous VEGF₁₆₅b, thereby preventing or reducing VEGF₁₆₅b-mediatedvasoconstriction where vasoconstriction is associated withpre-eclampsia, and thus may be due to excess VEGF₁₆₅b expression.

The inhibitor may comprise an antibody according to a preceding aspectof the invention, for example, the inhibitor may be an exon 9 specificneutralising antibody.

Alternatively, the inhibitor may be a polynucleotide having acomplementary sequence to the sequence of an isolated polynucleotideaccording to a previous aspect of the invention.

Inhibitors or VEGF₁₆₅b may be identified in suitable screens in which acandidate compound is screened for its ability to inhibit VEGF₁₆₅b. Suchscreens may be-carried out in respect of a plurality of compounds, forexample, from a compound library.

An inhibitor identified using such a screen may be synthesised andformulated into a pharmaceutical composition for use.

The present invention also provides a method for preventing or reducingvasoconstriction in a mammalian patient comprising supplying to thepatient an inhibitor according to a preceding aspect of the invention.

The present invention also provides a use of an inhibitor according to aprevious aspect of the invention for the preparation of a pharmaceuticalcomposition for the treatment of vasoconstriction.

The invention also provides an assay for the specific detection ofVEGF₁₆₅b in a sample comprising carrying out a polymerase chain reactionon at least a portion of the sample, using an annealing temperature ofat least 59° C. and the following primer sequences: exon 4 (forwardprimer): GAGATGAGCTTCCTACAGCAC 9H (reverse primer):TTAAGCTTTCAGTCTTTCCTGGTGAGAGATCTGCAor variants thereof, wherein the variants retain the same annealingproperties with respect to the VEGF₁₆₅b nucleotide sequence as the aboveprimers.

This method allows the specific detection of VEGF₁₆₅b in a sample, evenwhen the sample contains both VEGF₁₆₅b and VEGF₁₆₅.

Preferably, an annealing temperature of 60° C. is used.

Preferably, the method can detect VEGF₁₆₅b in a sample containingVEGF₁₆₅ at a concentration of up to 100 times greater, and preferably upto 500 times greater, more preferably 1000 times greater than theconcentration of VEGF₁₆₅b in the sample.

A further aspect of the invention provides an isolated VEGF nucleotidesequence according to the present invention for use in the detection ofexon 9 containing VEGF isoforms by quantitative real-time PCR. A numberof technologies are available and known to those skilled in the art,these technologies would for example include but not be limited to:Taqman, Scorpion, Molecular Beacon (FRET).

In preceding aspects of the invention, VEGF₁₆₅b or its inhibitor ispreferably supplied to a patient by injection. Preferably, VEGF issupplied to a patient by intra-arterial, intravenous, intramuscular,intra-peritoneal or subcutaneous injection. However when localapplication is required a regional injection may be preferable, and thiscan be inferred from the site of the disease process eg intra-articularor intra-orbital injection. Alternatively, VEGF₁₆₅b or inhibitor thereofmay be supplied orally or topically. For example, in the treatment ofgingivitis, VEGF₁₆₅b could be supplied orally in the form of atoothpaste or mouthwash containing VEGF₁₆₅b. For psoriasis VEGF₁₆₅bcould be contained within an emollient cream, for herpes ocularinfection in the form of eye-drops or cream, for pulmonary lesions in anebulized aerosol.

The combination of VEGF₁₆₅b with other agents for simultaneousadministration to a mammalian patient via any of the aforementionedroutes in any clinical situation in which the simultaneousadministration may be deemed beneficial in the light of the poperties ofVEGF₁₆₅b and the other agent or agents. Specific combinations ofVEGF₁₆₅b and another agent or agents would be apparent to the skilledperson from the particular clinical situation involved. For example,such a combination may be the simultaneous administration in a cream oreyedrop of VEGF₁₆₅b and acyclovir or similar such agent, for thesimultaneous treatment of ocular herpes infection and the cornealangiogenesis that accompanies it. Another such example is thesimultaneous administration of VEGF₁₆₅b and an anti-inflammatory agentvia intrarticular injection in rheumatoid disease. Another such exampleis the simultaneous administration of VEGF₁₆₅b and a chemo orimmunotherapeutic agent to a tumour condition in a patient.

A further aspect of the invention provides an isolated chemical orbiological agent that can inhibit a switch of splicing from sequencesdescribed in the said invention to previously described sequences. Forexample, such an inhibitor could be used to prevent the splicing of asequence to exclude exon 9. For example, the splicing of mRNA to giveVEGF₁₆₅ (which includes exon 8 rather than exon 9) instead of VEGF₁₆₅b(which contains exon 9 rather than exon 8) could be prevented. Thiswould allow the treatment of VEGF₁₆₅-mediated conditions by supplying toa patient the agent that inhibits the switch in splicing from VEGF₁₆₅bto VEGF₁₆₅.

Embodiments of the present invention will now be described, by way ofexample only, and with reference to the following figures, in which:

FIG. 1 is a diagram showing the mRNA splicing and structure of the VEGFpre-mRNA and positions of relevant PCR primers EX7a, Ex7b, 3′UTR, V165Kand V165X;

FIGS. 2A and 2B are photographs of agarose gels containing bandscorresponding to PCR products FIG. 3A is a photograph of an agarose gelshowing the PCR products obtained using exon 7a primer and 3′ UTR primerand mRNA templates obtained from the opposite poles of four humannephrectomy specimens;

FIG. 3B is a box plot of the number of tissue samples with a bandcorresponding to VEGF₁₆₅b (black) or without VEGF₁₆₅b (stippled) inbenign and malignant tissues. FIG. 4A is the nucleotide sequence ofVEGF₁₆₅ and VEGF₁₆₅b cDNA. The 66 bp downstream of exon 7 are missingfrom VEGF₁₆₅b.

FIG. 4B is the exon structure of the C-terminal end of VEGF₁₆₅ andVEGF₁₆₅b. The 3′ UTR sequence of exon 8 contains a consensus intronicsequence for exon 9, a CT rich region and a CAG immediately prior to thesplice site. The nucleotide sequence results in an alternate 6 aminoacid C terminus. Capital letters are open reading frames, lower caseintrons or 3′UTR (italics, VEGF₁₆₅, bold, VEGF₁₆₅b).

FIG. 4C is the predicted amino acid sequence of VEGF₁₆₅ compared toVEGF₁₆₅b. The 6 alternative amino acids result in a different C-terminalstructure of the VEGF likely to affect receptor activation, but notreceptor binding or dimerisation. The Cys is replaced with a Ser and theC terminal amino acids are a basic (underlined) and an acidic (italics)moiety instead of two acidic ones. Therefore the net charge on this endof the molecule will be altered.

FIG. 5A is an agarose gel showing the ˜700 bp PCR product of V165K andV165X;

FIG. 5B is an SDS gel showing the products of the nested PCR using theexcised band as a template and exon7 and 3′UTR as primer pairs.

FIG. 6A shows the effect of VEGF₁₆₅b on VEGF₁₆₅ stimulated HUVECproliferation;

FIG. 6B shows dose response studies of the above effect.

FIG. 6C demonstrates the effect of VEGF₁₆₅b on VEGF₁₆₅ and FGFstimulated HUVEC ³H-thymidine incorporation;

FIG. 6D shows a dose response curve of the inhibition of cellproliferation compared to the log molar ratio of VEGF₁₆₅b to VEGF₁₆₅.

FIGS. 7A and 7B show the effect of VEGF₁₆₅ and VEGF₁₆₅b on the diameterof rat mesenteric arteries preconstricted with phenylephrine;

FIG. 8 is a photograph of an agarose gel showing the PCR productsobtained using exon 9 specific primers and VEGF₁₆₅ and VEGF₁₆₅btemplates and annealing temperatures of 58° C. and 60° C. and a range ofMgCl₂ concentrations;

FIG. 9A is a photograph of an agarose gel showing the PCR productsobtained using VEGF₁₆₅ and VEGF₁₆₅b templates and an annealingtemperature of 60° C. in PCR competition assays. Lanes 2 and 6 indicatethe reactions in which the primers detected VEGF₁₆₅b in preference toVEGF₁₆₅ when VEGF₁₆₅ was present at 1000× the concentration of VEGF₁₆₅b;

FIG. 9B is a photograph of a Western blot showing expression ofVEGF₁₆₅b, VEGF₁₆₅ produced by transfected HEK293 cells and commercialVEGF (Peprotech, N.J., USA);

FIGS. 10A and 10B show photographs of agarose gels containing PCRproducts of a tissue screen using primers which detect exon 8 and exon 9containing (10A) isoforms and Exon 9 specific isoforms (FIG. 10B);

FIG. 11 shows the effect of VEGF₁₆₅b on VEGF₁₆₅-mediated HUVECmigration.

FIG. 12 summarises the sequence identity of exon 9 in different species.

FIG. 13 shows PCR products using exon 9 (13 a) or exon 8 (13 b)specificprimers showing VEGF₁₆₅b mRNA expression in normal, osteoarthritic andrheumatoid synovium, and, (13 c) their relative expression.

FIGS. 14A and 14B show the expression of exon 8 and exon 9 containingVEGF isoforms in individual human pancreatic islets.

FIGS. 15 details the change in VEGF₁₆₅b expression in malignant versusbenign Trans-urethral resection of prostate curettings;

FIG. 16 is a photograph of a gel showing PCR products from archivalradical prostatectomy samples; and

FIG. 17 shows the VEGF concentration in media taken from human embryonickidney cells (HEK293) transfected with 2 μg VEGF₁₆₅b cDNA (in expressionvector pcDNA3) alone or with increasing amounts of double stranded siRNAdirected across the exon 7-exon 9 splice site.

EXAMPLE 1

Immediately after nephrectomy, cubes of tissue were harvested from theperiphery of a macroscopic human kidney tumour and from the benigncortical tissue of the opposite pole of the kidney. 100-200 mg of tissuewas homogenised in Trizol reagent and mRNA extracted by addition ofchloroform. The tissue was centrifuged and the aqueous layer removedinto isopropanol. The mRNA was pelleted and resuspended in 20 μl DEPCtreated water. 4 μl of RNA was reverse transcribed using MMLV RT andpolydT as a primer. The cDNA was then amplified using 1 μM of the 3′UTRprimer (ATGGATCCGTATCAGTCTTTCCT) and 1 μM of primers for either exon 7a(GTAAGCTTGTACAAGATCCGCAGACG), or exon 7b (GGCAGCTTGAGTTAAACGAACG), 1.2mM M_(g)Cl₂, 2 mM dNTPs, and 1 unit of Taq polymerase (Abgene) in itsbuffer. Reactions were cycled 35 times, denaturing at 96° C. for 30seconds, annealing at 55° C. for 30 seconds, and extending at 72° C. for60 seconds. The position of annealing of the primers used to the VEGFisoforms is shown in FIG. 1.

PCR products were run on a 3% agarose gel containing 0.5 μg/ml ethidiumbromide and visualised under a UV transilluminator. Exon 7a and 3′ UTRprimers give a product consistent with VEGF₁₄₈ of 164 bp, and oneconsistent with VEGF_(165, 183, 189) and ₂₀₆ of 199 bp (see FIG. 2A).Exon 7b and 3′ UTR primers give a product consistent withVEGF_(165, 183, 189,) and ₂₀₆ of 130 bp, but no product corresponding toVEGF₁₄₈ (see FIG. 2B). The band at 164 bp was excised from the gel underUV transillumination and the DNA extracted using Qiaex (Qiagen). The DNAwas then digested with BamHl and HinDIII and ligated intopBluescriptKSII (Stratagene). Ligations were transformed intosupercompetent XL-1 Blue E. coli (Stratagene), and grown on ampicillinresistant LB agar plates. Colonies were amplified and the plasmid DNApurified using Qiagen columns. The DNA was then sequenced using T7 andT3 sequencing primers by fluorescent dideoxy termination sequencing(ABI370). Sequences were analysed by automated fluorescentchromatography, and the sequence checked against the chromatograph byeye.

The resulting sequence is shown in SEQ.ID No. 4. In four of the samplesthe normal tissue had significantly more ˜150 bp product than ˜200 bpproduct. One of these samples was used to confirm the full length of thenew isoform. The entire length of the product was amplified by RT-PCRusing primers downstream of the original 3′UTR primer (V165X, AAT CTAGAC CTC TTC CTT CAT TTC AGG) engineered with an xbal site, and a primercomplementary to the translation intitiation site of the other isoformsof VEGF (V165K, CCG GTA CCC CAT GAA CTT TCT GC) engineered with a Kpn1site and PCR conditions as previously described.

PCR was carried out using exon 7b and 3′ UTR primers or exon 7a and 3′UTR primers and a selection of mRNAs purified from the opposite poles offour human kidneys as template RNA. By using opposite poles of thekidneys, both malignant tissue (at one pole) and benign tissue (at theother pole) samples were obtained from each of the four human kidneysstudied. The PCR used the same reaction conditions as previouslydescribed.

A sample of the results can be seen in FIG. 3. Of eighteen kidneys, 17had detectable levels of expression of a VEGF isoform consistent withVEGF₁₄₈B in the benign tissue, whereas only 4 of the 18 kidney samples(p<0.001, Fishers Exact Test) had detectable levels of expression in themalignant tissue. Subsequent sequencing of this VEGF isoform revealed anunexpected 3′ sequence (FIG. 4). The sequence indicated that the mRNAwas spliced from the 3′ end of exon 7 into the 3′ untranslated region ofVEGF₁₆₅ mRNA+44 bp from the end of exon 8. This splice site has the samefirst two nucleotides as exon 8, but a different 3′ sequence (see FIG.4A), which has been designated exon9. The intronic region between exon 7and exon 9 has an intronic consensus sequence of 5′GT . . . CAG3′ and ahigh CT rich region 6-24 bp prior to the 5′ end of exon 9. PCR of thefull length product using primers V165K and V165X resulted in one strongband at approximately 670 bp. Further, nested PCR using the 3′UTR and 7aprimers described above resulted in a strong band at 133 bp, confirmingthat the full length was VEGF₁₆₅b. Cloning of the full length of thissequence and subsequent sequence analysis using one of the clones(clone 1) revealed a 663 nucleotide sequence with a single open readingframe encoding a peptide 191 amino acids long. This peptide consisted ofthe same N terminal 185 amino acids as VEGF₁₆₅ (i.e. a 26 amino acidsignal sequence followed by 159 amino acids corresponding to exons 1, 2,3, 4, 5 and 7). However, the C terminal 6 amino acids were not the sameas exon 8 (see FIG. 4B). The six amino acids that this new exon codesfor are Ser-Leu-Thr-Arg-Lys-Asp, followed by a stop codon, TGA. Sincethis splice variant would code for a mature 165 amino acid polypeptide(after cleavage of the signal sequence) with 96.4% identity withVEGF₁₆₅, this isoform has been designated VEGF₁₆₅b.

The region between exon 7 and exon 9 that has been spliced out inVEGF₁₆₅b includes 66 bp making up exon 8 and the first 44 bp of the3′UTR of VEGF₁₆₅. Therefore, the PCR product resulting from VEGF₁₆₅b andexon 7+3′UTR primers is 66 bp shorter than VEGF₁₆₅, hence the lower bandseen on the gel in FIG. 3 a which corresponds to the new VEGF isoform.

VEGF₁₄₈ lacks 35 nucleotides of exon 7, but retains the exon 8nucleotide sequence (which remains untranslated due to the introductionof a stop codon due to a frame shift) and the untranslated regionbetween exons 8 and 9, and the UTR 3′ to exon 9. Therefore, the PCRproduct of VEGF₁₄₈ and exon 7+3′UTR primers would only be 31 bp shorterthan the VEGF₁₆₅b PCR product using the same primers, hence the originalobservation that the lower band on the gel is consistent with VEGF148.However, the lower band, in fact, corresponds to new isoform VEGF₁₆₅b.

Exons 8 and 9 both code for 6 amino acids and a stop codon. The aminoacid sequences are completely different. Exon 8 encodes for CDKPRR. Thecysteine forms a disulphide bond with Cys146 in exon 7. This results inthe carboxy terminus of VEGF₁₆₅ being held close to the receptor bindingdomain in exon 3 in the three dimensional structure of VEGF. In additionthe proline inserts a kink in the amino acid backbone of the molecule tofurther appose the final three amino acids at the receptor bindingdomain. Finally the two terminal amino acids are highly positivelycharged arginine residues. These charged residues will be held veryclose to the receptor binding and in the crystal structure of thereceptor ligand complex appear to be in a position to interact with thereceptor. This, indicates that these amino acids may be necessary forreceptor stimulation. Exon 9 on the other hand codes for SLTRKD and haslost the cysteine residue and therefore the carboxy terminus may not beheld into the receptor binding site. In addition there is no prolinekink, and therefore the terminal two amino acids may not be able tointeract with the receptor. It may therefore be possible that VEGF₁₆₅bwill bind to the VEGF receptor but not activate it. This theory arisessince VEGF₁₆₅b contains all the elements required for i) efficientdimerisation B CyS² and Cys⁴ in exon 3 (21); ii) receptor binding B forVEGFR1 Asp63, Glu64, Glu67 in exon 3, for VEGFR2 Arg82, Lys84, His 86 inexon 4, and for neuropilin-1 Cys136 to Cys158 in exon 7. This isoformsubstitutes exon 9 for exon 8 and therefore has no Cys159 which normallybinds to Cys146 in exon 7, and will therefore be likely to affect thefolding and tertiary structure of the VEGF molecule.

The fact that this isoform has been found specifically in the kidney isparticularly interesting as the kidney, and in particular the glomerulushas been shown to produce high levels of VEGF. This has always beenassumed to be VEGF₁₆₅, since it is detected by antibodies to VEGF₁₆₅, insitu hybridisation using probes to VEGF₁₆₅, PCR using primers specificto VEGF₁₆₅ and so on. However, in almost all these cases the detectiontechniques used would not distinguish between VEGF₁₆₅ and VEGF₁₆₅b. Theonly reason that VEGF₁₆₅b has been detected now is that an attempt wasbeing made to examine VEGF₁₄₈ expression in this tissue. It isinteresting that, despite high levels of VEGF produced by podocytes inthe glomerulus, glomerular endothelial cells, which do express VEGFreceptors, are not of an angiogenic phenotype. Endothelial cell turnoverin the glomerulus is low, as in other parts of the body, and there is noovert angiogenesis occurring. This apparent paradox—high VEGF expressionbut low angiogenesis—may be explained by VEGF₁₆₅b. Kidney tumours mustgrow in an environment in which VEGF is highly expressed under normalconditions, and yet angiogenesis is prevented. Therefore tumours need toovercome some endogenous anti-angiogenic process in order to grow. IfVEGF₁₆₅b is inhibitory then the down regulation of VEGF₁₆₅b by tumours,as shown here, would be necessary for tumours to switch angiogenesis onin this tissue. Furthermore, the high production of VEGF by theglomerulus may actually be VEGF₁₆₅b rather than VEGF₁₆₅, and thereforethis would explain why angiogenesis is not observed.

A further clone (clone 2) containing the full length PCR product(obtained using primers V165K and V165X) was selected and sequenced.Surprisingly, although this sequence contained the same C terminal 6amino acids as clone 1, indicating the presence of exon9, the fulllength of the cloned sequence was longer than that of the sequence ofclone 1, i.e. longer than 670 bp and therefore not consistent withVEGF₁₆₅b. In fact, the sequence length was found to be slightly over 700bp, which is more consistent with VEGF₁₈₃b (see FIG. 5). FIG. 5B showsthe products of nested PCR using the excised ˜700 bp band as templateand exon 7 and 3′UTR as primer pairs.

It therefore seems that a family of VEGF isoforms may exist which possesexon 9 in place of exon 8. The existence of VEGF₁₆₅b has beendemonstrated in the example provided herein and and evidence forVEGF₁₈₃b is presented. However, it is likely that similar isoforms existwhich correspond to VEGF₁₂₁, VEF₁₄₅, VEGF₁₈₉, VEGF₂₀₆, designatedVEGF₁₂₁b, VEGF₁₄₅b, VEGF₁₈₉b and VEGF₂₀₆b. These isoforms which lackexon 8 are all expected to demonstrate anti-angiogenic activity. TABLE 1Expected PCR product size for V165K and V165X primers for the differentsplice variants of VEGF. No of amino acids 121 145 148 165 183 189 206No of VEGF_(XXX) 597 669 694 729 783 801 852 nucleotide VEGF_(XXX)b 531603 628 663 717 735 786 s3′

EXAMPLE 2

In separate transfections, full length VEGF₁₆₅b cDNA and full lengthVEGF₁₆₅ cDNA were cloned into pcDNA3 using standard methodology, andthen transfected into HEK 293 cells and a stable cell line generatedusing Geneticin selection. Confluent cells were incubated in basal M200endothelial cell medium for 48 hours containing neither serum norGeneticin, and the conditioned media assayed for VEGF concentrationusing a pan VEGF ELISA (R & D Systems). Control (mock) conditioned media(pcDNA3-CM) was collected in the same way from HEK293 cells transfectedwith pcDNA3 only.

Freshly isolated HUVECs incubated in 0.1% serum M200 overnight wereincubated with either pcDNA3-CM, VEGF₁₆₅-CM (adjusted to 100 ng/mlVEGF₁₆₅ with pcDNA3-CM), VEGF₁₆₅b-CM (adjusted to 100 ng/ml VEGF₁₆₅bwith pcDNA3-CM) or a mixture of VEGF₁₆₅b-CM and VEGF₁₆₅-CM (adjusted to100 ng/ml VEGF₁₆₅b and 100 ng/ml VEGF₁₆₅ with pcDNA3-CM), and then 37kBq of ³H-Thymidine (Amersham Pharmacia) added. After 4 hrs the cellswere washed, trypsinised, cell number counted on a hemocytometer andradioactivity measured on a beta counter (LKB-1217). Incorporation wascalculated as counts per cell. Dose response curves were carried out ina similar manner, except that VEGF₁₆₅b-CM was diluted with pcDNA3-CM and50ng/ml VEGF₁₆₅ (Peprotech, N.J.) added. Proliferation index wascalculated as ³H-thymidine incorporation of VEGF₁₆₅ treated cellscompared to mean incorporation into cells with no VEGF₁₆₅ treatment.

To determine the functional effect of VEGF₁₆₅b we measured endothelialcell proliferation by determination of ³H-thymidine incorporation percell number during incubation with conditioned media from transfectedcells (FIG. 6). The full-length cDNA generated by PCR was cloned into anexpression vector (pcDNA3-VEGF₁₆₅b), as was full length VEGF₁₆₅(pcDNA3-VEGF₁₆₅) and each transfected into HEK293 cells. The VEGFconcentration of cell conditioned media (VEGF₁₆₅b-CM), as determined byELISA using pan VEGF antibodies, ranged from 80-400 ng/ml VEGF.Conditioned media from cells transfected with pcDNA3-VEGF₁₆₅(VEGF₁₆₅-CM) had a VEGF concentration of 100-260 ng/ml. Media from cellstransfected with pcDNA3 alone (pcDNA3-CM) contained <62.5 pg/ml VEGF(the minimum detection limit of the ELISA). HUVECs were incubated inVEGF₁₆₅-CM adjusted to 100 ng/ml (with pcDNA3-CM) and this resulted in asignificant 283±43% increase in thymidine incorporation per endothelialcell compared to pcDNA3-CM alone (P<0.01, ANOVA, FIG. 6 a). VEGF₁₆₅b-CMcontaining 100 ng/ml VEGF₁₆₅b did not stimulate HUVEC proliferation(165±27% of pcDNA3-CM, no significant increase, but significantly lessthan VEGF₁₆₅-CM, p<0.05). Furthermore, there was no increase inendothelial cell proliferation when cells were incubated in acombination of both VEGF₁₆₅b-CM and VEGF₁₆₅-CM containing 100 ng/ml ofeach VEGF isoform (150±18% of pcDNA3-CM, not significantly differentfrom pcDNA3-CM, but significantly lower than VEGF₁₆₅-CM, p<0.05).Therefore VEGF₁₆₅b did not stimulate HUVEC proliferation, and moreover,significantly inhibited VEGF₁₆₅ stimulated proliferation. Furthermorewhen endothelial cells were incubated in CM containing increasingconcentrations of VEGF₁₆₅b there was a dose dependent inhibition of³H-thymidine incorporation stimulated by commercially available VEGF₁₆₅(FIG. 6 b), with a molar ratio IC₅₀ of 0.94 (FIG. 6 d) (i.e. equimolarinhibition). Additionally, VEGF₁₆₅b-CM did not affect FGF mediatedproliferation (FIG. 6 c).

EXAMPLE 3

The effect of VEGF₁₆₅b on VEGF₁₆₅-mediated vasodilatation was determinedas detailed below.

3^(rd) order superior mesenteric arteries were dissected from 200-300 gmale Wistar rats (sacrificed by stunning and cervical dislocation) andleak-free segments mounted in an arteriograph at 80 mmHg, in M200media10 mm acetylcholine (Ach) maximally dilated all arteries used inthis example, demonstrating the presence of an intact endothelium.

The rat mesenteric arteries were preconstricted with 0.6-1 μmphenylephrine (PE) applied in the superfusate (see FIG. 7A). Ach, CM andVEGF isoforms (all dialysed against rat ringer solution using 3500 MWdialysis tubing) were then applied to the lumen of the artery using anadaptation of the Halpern pressure myograph technique as described inDoughty, J. M. et al (1999) Am J Physiol 276, 1107-12. The concentrationof VEGF in the CM after dialysis was determined by ELISA. All data areexpressed as mean values ± s.e. mean for 4 experiments. Statisticalsignificance was tested using ANOVA and Student Newmann Keuls post-hoctest.

We measured the effects of VEGF₁₆₅b on vasodilatation to determinewhether VEGF₁₆₅b could inhibit the effects of VEGF₁₆₅ on intact vessels.Luminal perfusion of isolated pressurized rat mesenteric arteries invitro with pc-DNA3-CM resulted in no change in vessel diameter (FIG. 7).Perfusion of the same arteries with dialyzed VEGF₁₆₅b-CM (40 ng/ml) didnot affect the diameter of the arteries either. Perfusion with dialyzedCM containing 20 ng/ml VEGF₁₆₅ resulted in significant vasodilatation,but this vasodilatation was abolished when perfused with VEGF₁₆₅b-CM andVEGF₁₆₅ (40 ng/ml VEGF₁₆₅b, 20 ng/ml VEGF165). Therefore VEGF₁₆₅b doesnot stimulate vasodilatation, and is also capable of inhibitingVEGF₁₆₅-mediated vasodilatation.

EXAMPLE 4

As mentioned earlier, the kidney, and in particular the glomerulus, hasbeen shown to produce high levels of VEGF. This has always been assumedto be VEGF₁₆₅, since it is detected by antibodies to VEGF₁₆₅, or by insitu hybridisation using probes to VEGF₁₆₅, or by PCR using primersspecific to VEGF₁₆₅.

However, in all of these cases, the detection techniques used would notdistinguish between VEGF₁₆₅ and VEGF₁₆₅b.

A method of detection of VEGF₁₆₅b mRNA independently of VEGF₁₆₅ mRNA hasbeen developed and is detailed below (FIGS. 8 and 9A).

PCR was carried out using vectors containing VEGF₁₆₅ and VEGF₁₆₅b,respectively, and the following primers: Forward primer Exon-4GAGATGAGCTTCCTACAGCAC Reverse Primer 9HTTAAGCTTTCAGTCTTTCCTGGTGAGAGATCTGCA

The annealing and denaturing steps were carried out for 30 seconds anddenaturing was carried out at 94° C. 1 minute periods of extension werecarried out at 72° C.

As can be seen from FIG. 8, when an annealing temperature of 58° C. isused, PCR products are obtained for both VEGF₁₆₅ and VEGF₁₆₅b. However,when an annealing temperature of 60° C. is used, there is nocross-reactivity and no PCR product corresponding to VEGF₁₆₅ isobtained.

Competitive PCR studies were carried out using the PCR primers andconditions mentioned above, with the annealing step being carried out at60° C. The samples used contained VEGF₁₆₅ and VEGF₁₆₅b at variousrelative concentrations, as shown in FIG. 9 a. FIG. 9 a demonstratesthat, even when VEGF₁₆₅ is present in the sample at 1000 times theconcentration at which VEGF₁₆₅b is present, the only PCR productobtained corresponds to amplification of VEGF₁₆₅b and not VEGF₁₆₅.

Therefore, the above primers can be used to detect VEGF₁₆₅ and VEGF₁₆₅bin a sample. Further, by altering the temperature at which the PCRannealing step is carried out, the same primers can be used tospecifically detect the presence of VEGF₁₆₅b in a sample containing bothVEGF₁₆₅ and VEGF₁₆₅b.

EXAMPLE 5

Full length VEGF₁₆₅b or VEGF₁₆₅ (generated by PCR from tissue) wascloned into the expression vector pcDNA₃ using standard methodology, andthen transfected into HEK 293 cells and a stable cell line generatedusing Geneticin selection. Confluent cells were incubated in basal M200endothelial cell medium for 48 hours, containing neither serum norGeneticin, and the conditioned media assayed for VEGF concentrationusing a pan VEGF ELISA(R&D). Control conditioned media (pcDNA3-CM) wascollected in the same way from HEK293 cells stably transfected withpcDNA₃. FIG. 9 b shows a Western blot of conditioned media from VEGF₁₆₅btransfected cells (VEGF165b-CM) and VEGF₁₆₅ transfected cells(VEGF₁₆₅-CM). VEGF₁₆₅b was the same molecular weight as VEGF₁₆₅, andboth isoforms correspond to previously published molecular weights forVEGF₁₆₅ (Ferrara N et al Biophys. Res. Comm. 1989; 161: 851-8). The blotalso confirms that most of the VEGF₁₆₅ and VEGF₁₆₅b made in HEK cellshad a slightly greater molecular weight than commercially availableVEGF₁₆₅ (23 kDa compared to 18 kDa). This is probably due to the factthat commercially available VEGF₁₆₅ is not glycosylated. There was someVEGF₁₆₅b and VEGF₁₆₅ in the conditioned media that appeared to bede-glycosylated, but this was a small fraction of the total VEGF.

EXAMPLE 6

mRNA from sixteen different tissues was reverse transcribed andamplified using exon 7 and 3′UTR primers. PCR products of lengthsconsistent with expression of exon 9 containing isoforms were clearlydetected in umbilical cord, cerebrum, aorta, prostate, pituitary, lung,skeletal muscle and placental tissue as well as kidney (see FIG. 10A).Fainter bands were also seen in colon, skin, bladder and spinal cord. Nosignificant exon 9-containing isoforms were detected in thehypothalamus, inferior vena cava (IVC), or liver. Subsequent PCR usingexon 9-specific primers confirmed the distribution of expression in thedifferent tissues (see FIG. 10B), but in this case expression wasdetected in liver, and expression in the pituitary was marginal.Interestingly in aorta, prostate and umbilical cord a slightly longerband was seen. It is not clear whether these are additional exon9-containing isoforms such as VEGF₁₈₃b and/or VEGF₁₈₉b.

EXAMPLE 7

In order to assess the functional properties of VEGF₁₆₅b on endothelialcell migration, migration assays were performed in a modified 24-wellBoyden chamber containing collagen coated polycarbonate filter inserts(8 μm pore; Millipore). The filters were placed in 24 well platescontaining 0.5 ml per well of either 1) VEGF₁₆₅-CM containing 33 ng/mlVEGF₁₆₅; 2) VEGF₁₆₅b-CM containing 33 ng/ml VEGF₁₆₅b; 3) VEGF₁₆₅-CM andVEGF₁₆₅b-CM (33 ng/ml of each isoform); or 4) pcDNA₃-CM CM. HUVECs weresuspended in serum free medium and 25,000 cells added to the upperchamber of each well. The plate was incubated for 6 h to allowmigration, media removed and both chambers washed with PBS (×2). 0.2mg/ml Thiazolyl Blue (MTT) in media was then added to both chambers andincubated for 3 h at 37° C. The media was removed and the chambers werewashed with PBS (×2). Non-migrated cell crystals in the upper chamber(stained blue) were removed with a cotton swab, which was placed in 1 mlof Dimethyl Sulphoxide (DMSO) to dissolve the MTT product. Migratorycell crystals (on the underside of the insert) were also dissolved inMTT. The samples were left overnight to permit complete solution of theproduct. The absorbance of soluble MTT was determined at a wavelength of570 nm using a spectrophotometer. The percentage migration was thencalculated from the intensity of the lower well as a percentage of thetotal intensity of both wells. Assays were run in sextuplet.

HUVECs were incubated in VEGF₁₆₅-CM (33 ng/ml VEGF₁₆₅) and this resultedin a significant 24±3% increase in migration of endothelial cellscompared to pcDNA3-CM alone (P<0.01, ANOVA)(FIG. 11). VEGF₁₆₅b -CMcontaining 33 ng/ml VEGF₁₆₅b did not stimulate migration (−3±2.6%compared to pcDNA3-CM, not significant). Furthermore, there was noincrease in migration when cells were incubated in a combination of bothVEGF₁₆₅b-CM and VEGF₁₆₅-CM containing 33 ng/ml of each isoform (9.9±5.8%compared to pcDNA3-CM). Therefore VEGF₁₆₅b did not stimulate migration,and again significantly inhibited VEGF₁₆₅-stimulated migration (P<0.001,ANOVA).

EXAMPLE 8

A search of the nucleotide database provides interesting information onthe conservation of the 3′ untranslated region (3′UTR). The entire 3′UTR from the end of exon 8 is 100% conserved between human and macaque.In other mammalian species there is relatively good conservation of thesupposed 3′ UTR terminal to exon 8 (FIG. 12). In fact, in the cow themRNA has >95% identity in the 66 bases 3′ to the stop codon of exon 8,and exon 9 is >90% identical. However, this identity breaks downimmediately after the exon 9 stop codon (see FIG. 12). There issignificantly less identity in the 22 bases after this stop codon (53%)than that in exon 9 (91%). This pattern is also evident in mouse wherethe exon 9 containing sequence is 86% identical to human, but only 23%identical in the 22 base pairs immediately after the exon 9 stop codon(see FIG. 12). Interestingly the mouse sequence predicts an exon 9 of 7amino acids of the sequence PLTGKTD, compared to SLTRKD in the human andmacaque and RLTRKD in the cow. Therefore 4 out of six amino acids areconserved [XLTXK(X)D] and this appears to have been brought about by adouble mutation—an adenosine insertion in the human at nucleotide 10(mouse) and a cytosine to thymidine mutation at nucleotide 19 (mouse)that rescues the stop codon (see FIG. 12). This is indirect evidence forfunctional relevance of the splice site. Interestingly it is onlyconserved in mammals, not in birds or fish.

EXAMPLE 9

VEGF has been shown to be massively upregulated in arthritis,particularly in synovial fibroblasts, macrophages and synovial liningcells (Nagashima M et al, 1995; J Rheumatol 22: 1624-30). Furthermorethere is now mounting functional evidence that inhibition ofangiogenesis, both non-specifically with pharmacological anti-angiogenicagents (Oliver S et al Cell Immunol 1994; 157: 291-9; Oliver S. et alCell Immunol 1995; 166: 196-206), and specifically with anti-VEGFagents, ameliorates the joint lesions in well characterised animalmodels of arthritis (Miolta J et al Lab Invest 2000; 80: 1195-205; SoneH et al Biochem Biophys Res Commun 2001; 281: 562-8). Using an RT-PCRprotocol described under example 6, VEGF₁₆₅b expression has beenidentified in human synovium (taken at operation from a patient whosuffered a fractured neck of femur secondary to osteoporosis). VEGF₁₆₅bexpression was shown to be present in normal human synovium collectedfrom the Bristol University Anatomy Department. In order to obtainpreliminary data on the expression of VEGF₁₆₅b mRNA in normal andarthritic tissue, RT-PCR was carried out on two previously availablesamples of osteo tissue and two of rheumatoid tissue (see FIG. 13).Interestingly VEGF₁₆₅b was found in three of these four samples, but ata lower level than VEGF₁₆₅. This was in stark contrast to normal tissuewhich had at least as much VEGF₁₆₅b as VEGF₁₆₅. The ratio of VEGF₁₆₅b toVEGF₁₆₅ mRNA in synovium was therefore, lower in rheumatoid arthritisthan osteoarthritis, and lower in osteoarthritis than normal tissue.This suggests that in arthritis the anti-angiogenic properties ofVEGF₁₆₅b also are reduced.

EXAMPLE 10

Human islet transplantation offers a unique therapeutic option in themanagement of the metabolic and vascular sequelae of diabetes mellitus.However animal models and clinical experience suggest that transplantedislet function is not predictable in an individual recipient. It hasbeen suggested that the initial function of transplanted islets isdependent on their ability to access the recipient vascular system bythe stimulation of microvascular angiogenesis.

Human islets were purified by collagenase digestion (Liberase HI, RocheDiagnostics) and continuous density gradient centrifugation on a Cobe2991 cell separator. Individual islets were then collected in 20 μldiethylpyrocarbonate (depc) H₂O in separate tubes. Each sample washomogenized in Trizol reagent and mRNA was extracted according to themanufacturers' instructions. mRNA was reverse transcribed using polyd(T)as a primer and ExpandRT (Roche). Exon 8 and exon 9 containing VEGFisoforms were studied in 11 individual islets. A heterogeneity ofexpression of both VEGF families was identified (see FIG. 14). It isbelieved that the balance of pro- and anti-angiogenic VEGF isoformexpression by transplanted islets may determine the survival ofindividual islets and hence the overall efficiency of the graft.Inhibition of the activity of anti-angiogenic VEGF₁₆₅b may thereforeenhance graft survival and function.

EXAMPLE 11

In order to assess if VEGF₁₆₅b expression is altered in cancers otherthan renal cancer, VEGF₁₆₅b expression was studied in prostate.

VEGF₁₆₅b expression was studied in trans-urethral prostatectomycurettings using the PCR protocol described under example 6 (Exonspecific primers). VEGF₁₆₅b was present in significantly less malignantsamples compared to benign (FIG. 15).

In addition, we studied VEGF₁₆₅b expression in archival radicalprostatectomy samples to determine the expression of VEGF165b in theearly stages of prostate cancer, i.e. in prostatic intra-epithelialneoplasia (PIN)(see FIG. 16). Significant down regulation of VEGF165bmRNA was seen in PIN lending credence to the hypothesis that theangiogenic switch in prostate cancer may result from an imbalance of proand anti-angiogenic VEGF isoforms. The template mRNA was obtained fromarchival tissue using a standard protocol (Krafft A E I et al. (1997);2(3); 217-230).

EXAMPLE 12

To assess the possibility of specifically inhibiting the VEGF₁₆₅bisoform, small interference RNAs to the exon7-exon9 boundary weredeveloped.

siRNA probes may be made according to the art from primers for exampleof the sequence: T7siRNA165bsR 5′ AGAGATCTGCAAGTACGTTCTATAGTGAGTCGTATTA3′ T7siRNA165basR 5′ ACGAACGTACTTGCAGATCTCTATAGTGAGTCGTATTA 3′siRNA165bF 5′ TAATACGACTCACTATAG 3′

These are T7 small interfering RNA for VEGF₁₆₅b sense reverse primer, T7small interfering RNA for VEGF₁₆₅b antisense reverse primer and smallinterference RNA for VEGF₁₆₅b forward primer, respectively.

Production of double stranded DNA duplexes from T7siRNA165bsR andT7siRNA165bF provide templates for sense RNA, and production of doublestranded DNA duplexes from T7siRNA165basR and siRNA165bF providetemplates for antisense RNA.

Annealing of these two RNAs results in the production of a doublestranded siRNA duplex with overhang of the sequence: 5′ GAACGUACUUGCAGAUCUCUC 3′     |||||||||||||||||||3′UGCUUGCAUGAACGUCUAGAG   5′

This double stranded siRNA duplex, when transfected into cells, willreduce VEGF₁₆₅b production, as shown in FIG. 17. Amino acid sequence ofExon 9 SLTRKD SEQ. ID. No 1 Nucleotide sequence of Exon 9ATCTCTCACCAGGAAAGACTGA SEQ. ID. No 2 Amino acid sequence of VEGF₁₆₅bMNFLLSWVHW SLALLLYLHH AKWSQAAPMA EGGGQNHHEV SEQ. ID. No 3VKFMDVYQRSYCHPIETLVD IFQEYPDEIE YIFKPSCVPL MRCGGCCNDEGLECVPTEESNITMQIMRIK PHQGQHIGEM SFLQHNKCEC RPKKDPARQENPCGPCSERRKHLFVQDPQT CKCSCKNTDS RCKARQLELN ERTCR SLTRK DS Nucleotidesequence of VEGF₁₆₅b ATGA ACTTTCTGCT GTCTTGGGTG CATTGGAGCC TTGCCTTGCTSEQ. ID. No 4 GCTCTACCTC CACCATGCCA AGTGGTCCCA GGCTGCACCC ATGGCAGAAGGAGGAGGGCA GAATCATCAC GAAGTGGTGA AGTTCATGGA TGTCTATCAG CGCAGCTACTGCCATCCAAT CGAGACCCTG GTGGACATCT TCCAGGAGTA CCCTGATGAG ATCGAGTACATCTTCAAGCC ATCCTGTGTG CCCCTGATGC GATGCGGGGG CTGCTGCAAT GACGAGGGCCTGGAGTGTGT GCCCACTGAG GAGTCCAACA TCAGCATGCA GATTATGCGG ATCAAACCTCACCAAGGCCA GCACATAGGA GAGATGAGCT TCCTACAGCA CAACAAATGT GAATGCAGACCAAAGAAAGA TAGAGCAAGA CAAGAAAATC CCTGTGGGCC TTGCTCAGAG CGGAGAAAGCATTTGTTTGT ACAAGATCCG CAGACGTGTA AATGTTCCTG CAAAAACACA GACTCGCGTTGCAAGGCGAG GCAGCTTGAG TTAAACGAAC GTACTTGCAG ATCT CTCACCAGGA AAGACTGA

1. An isolated VEGF polypeptide having anti-angiogenic activity, saidpolypeptide including the amino acid sequence of SEQ.ID NO.1, orvariants thereof.
 2. An isolated VEGF polypeptide having anti-angiogenicactivity, said polypeptide having at least 66% identity to the aminoacid sequence of SEQ.ID NO.1.
 3. An isolated VEGF polypeptide havinganti-angiogenic activity, said polypeptide having at least 83% identityto the amino acid sequence of SEQ.ID NO.1.
 4. An isolated VEGFpolypeptide according to claim 1 wherein said polypeptide is capable ofbinding to endogenous VEGF and preventing or reducing stimulation ofmitosis.
 5. An isolated VEGF polypeptide according to claim 1, whereinsaid polypeptide comprises the amino acid sequence of SEQ. ID NO.3, orvariants thereof.
 6. An isolated nucleotide sequence capable of encodinga VEGF polypeptide according to claim
 1. 7. An isolated nucleotidesequence encoding a VEGF polypeptide having anti-angiogenic activity,the nucleotide sequence comprising the nucleotide sequence of SEQ.IDNO.2, or variants thereof.
 8. An isolated nucleotide sequence-encoding aVEGF polypeptide having anti-angiogenic activity, the nucleotidesequence comprising the nucleotide sequence of SEQ.ID NO.4, or variantsthereof.
 9. An isolated nucleotide sequence comprising a nucleotidesequence that hybridises under stringent conditions to the nucleotidesequence of SEQ.ID.NO:2, or variants thereof.
 10. An isolated nucleotidesequence according to claim 6 wherein the nucleotide sequence isselected from the group consisting of unprocessed RNA, ribozyme RNA,hairpin siRNA, siRNA, mRNA, cDNA, genomic DNA, B-DNA, E-DNA and Z-DNA.11. An isolated polypeptide according to claim 1, derived from amammalian sequence.
 12. An isolated polypeptide sequence or nucleotidesequence according to claim 11 wherein the mammalian sequence isselected from the group consisting of a primate, rodent, bovine sand aporcine sequence.
 13. (CANCEL)
 14. (CANCEL)
 15. (CANCEL)
 16. (CANCEL)17. (CANCEL)
 18. (CANCEL)
 19. A method for treating or preventingangiogenesis in a mammalian patient comprising supplying to the patienta polypeptide comprising the sequence of a VEGF polypeptide according toclaim
 1. 20. A method for treating or preventing angiogenesis in amammalian patient comprising supplying to the patient a polynucleotidecomprising a nucleotide sequence according to claim
 6. 21. An isolatedVEGF polypeptide according to claim 1, wherein said polypeptide iscapable of binding endogenous VEGF thereby preventing or reducingVEGF-mediated cell proliferation.
 22. A method for preventing orreducing VEGF-mediated cell proliferation in a mammalian patientcomprising supplying to the patient a polypeptide comprising thesequence of an isolated VEGF polypeptide according to claim
 1. 23. Amethod for preventing or reducing VEGF-mediated cell proliferation in amammalian patient comprising supplying to the patient a polynucleotidecomprising the sequence of an isolated nucleotide according to claim 6.24. (CANCEL)
 25. A pharmaceutical composition for the prevention orreduction of VEGF-mediated cell proliferation comprising the nucleotidesequence to claim 6 and a pharmaceutically acceptable diluent.
 26. Anisolated VEGF polypeptide according to claim 1 wherein said polypeptideis capable of binding endogenous VEGF thereby preventing or reducingVEGF₁₆₅-mediated vasodilatation.
 27. A method for preventing or reducingVEGF₁₆₅-mediated vasodilatation in a mammalian patient comprisingsupplying to the patient a polypeptide comprising the sequence of a VEGFpolypeptide according to claim
 26. 28. (CANCEL)
 29. (CANCEL)
 30. Amethod for preventing or reducing VEGF₁₆₅-mediated vasodilatation in amammalian patient comprising supplying to the patient a polynucleotidecomprising the sequence of an isolated nucleotide according to claim 6.31. (CANCEL)
 32. (CANCEL)
 33. A pharmaceutical composition comprising aVEGF polypeptide according to claim 1 and a pharmaceutically acceptablediluent.
 34. A pharmaceutical composition comprising a nucleotidesequence according to claim 12 and a pharmaceutically acceptablediluent.
 35. An expression vector comprising a nucleotide sequenceaccording to claim
 6. 36. A host cell comprising an expression vectoraccording to claim
 34. 37. (CANCEL)
 38. An antibody raised against apolypeptide comprising the sequence of a VEGF polypeptide according toclaim
 1. 39. An antibody produced against a polynucleotide comprising anucleotide sequence according to claim
 6. 40. An antibody according toclaim 38 wherein the antibody is a chimeric antibody.
 41. An antibodyaccording to claim 38 wherein the antibody is a humanised antibody. 42.A method of screening compounds to identify an agonist of theanti-angiogenic activity of a polypeptide according to claim 1 whereinsaid polypeptide and a labelled ligand of said polypeptide are incubatedin the presence and absence of a candidate compound, wherein increasedanti-angiogenic activity of said peptide in the presence of saidcandidate compound when compared to the anti-angiogenic activity in theabsence of said compound indicates that the candidate compound is anagonist.
 43. A method according to claim 42, comprising the steps of: a)incubating the polypeptide and the labeled ligand in the presence andabsence of a candidate compound; b) comparing the anti-angiogenicactivity of the peptide incubated in the presence of the candidatecompound with the anti-angiogenic activity of the peptide incubated inthe absence of the candidate compound; wherein increased anti-angiogenicactivity of the peptide incubated in the presence of the candidatecompound compared with the anti-angiogenic activity of the peptideincubated in the absence of the candidate compound indicates that thecandidate compound is an agonist.
 44. A method according to claim 43,further comprising the step of synthesising the agonist andincorporating the agonist into a pharmaceutical composition.
 45. Amethod of screening compounds to identify an antagonist of theanti-angiogenic activity of a polypeptide according to claim 1 whereinsaid polypeptide and a labelled ligand of said polypeptide are.incubated in the presence and absence of a candidate compound, whereindecreased anti-angiogenic activity of said peptide in the presence ofsaid candidate compound when compared to the anti-angiogenic activity inthe absence of said compound indicates that the candidate compound is anantagonist.
 46. A method according to claim 45, comprising the steps of:a) incubating the polypeptide and the labeled ligand in the presence andabsence of a candidate compound; b) comparing the anti-angiogenicactivity of the peptide incubated in the presence of the candidatecompound with the anti-angiogenic activity of the peptide incubated inthe absence of the candidate compound; wherein increased anti-angiogenicactivity of the peptide incubated in the presence of the candidatecompound compared with the anti-angiogenic activity of the peptideincubated in the absence of the candidate compound indicates that thecandidate compound is an antagonist.
 47. A method according to claim 46,further comprising the step of synthesising the antagonist andincorporating the antagonist into a pharmaceutical composition.
 48. Acompound identified by a method according to claim
 44. 49. A method fortreating a disorder comprising administering to a subject in needthereof an effective amount of the pharmaceutical composition of claim63, wherein the disorder is selected from the group consisting of tumourgrowth and metastasis, rheumatoid arthritis, atherosclerosis, neointimalhyperplasia, diabetic retinopathy and other complications of diabetes,trachoma, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, hemangiomas, immune rejection of transplantedcorneal tissue, corneal angiogenesis associated with ocular injury orinfection, vascular disease, obesity, psoriasis, arthritis, disease,obesity, psoriasis, arthritis, and gingival hypertrophy.
 50. A methodfor treating pre-eclampsia, comprising administering to a subject inneed thereof an effective amount of the pharmaceutical composition ofclaim
 63. 51. (CANCEL)
 52. A pharmaceutical composition for treating alack of VEGF₁₆₅-mediated vasodilatation comprising an inhibitor of aVEGF₁₆₅b polypeptide according to claim
 1. 53. A pharmaceuticalcomposition for treating a lack of VEGF₁₆₅-mediated vasodilatationcomprising an antibody according to claim
 38. 54. The pharmaceuticalcomposition according to claim 52 wherein the lack of vasodilatation isassociated with pre-eclampsia.
 55. A pharmaceutical composition fortreating a lack of VEGF₁₆₅-mediated vasodilatation comprising apolynucleotide having a complementary sequence to a nucleotide sequenceaccording to claim
 6. 56. (CANCEL)
 57. (CANCEL)
 58. A method forpreventing or ameliorating a lack of VEGF₁₆₅-mediated vasodilatation ina mammalian patient comprising supplying to the patient an inhibitoraccording to claim
 52. 59. A method according to claim 58 wherein thelack of vasodilatation is associated with pre-eclampsia.
 60. An assayfor the specific detection of VEGF₁₆₅b in a sample comprising carryingout a polymerase chain reaction on at least a portion of the sample,using an annealing temperature of at least 59° C. and the followingprimer sequences: exon 4 (forward primer): GAGATGAGCTTCCTACAGCAC 9H(reverse primer): TTAAGCTTTCAGTCTTTCCTGGTGAGAGATCTGCA

or variants thereof, wherein the variants retain the same annealingproperties with respect to the VEG₁₆₅ nucleotide sequence as the aboveprimers.
 61. An assay according to claim 60 wherein the primers arecombined with fluorescent detection probes for the detection of VEGF₁₆₅busing real time PCR protocols.
 62. An assay according to claim 61wherein the real time PCR protocols are selected from the groupconsisting of Molecular Beacon, FRET, TaqMan, and Scorpion.
 63. Apharmaceutical composition comprising the compound of claim 48 and apharmaceutically acceptable carrier.